Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense are protozoan parasites that cause African sleeping sickness in humans while Trypanosoma brucei brucei causes a wasting disease in cattle called Nagana. These parasites are morphologically indistinguishable and the only clear phenotypic distinction is the inability of T. b. brucei to infect humans. Innate protection against T. b. brucei infection is mediated by Trypanosome Lytic Factors (TLF) found in the serum of humans, apes and old-world monkeys. The active component of TLF is the primate specific haptoglobin related protein (HPR). This protein is the sole factor preventing infection of humans by T. b. brucei, thus severely restricting its host range. The human sleeping sickness trypanosomes, T. b. rhodesiense and T. b. gambiense, are able to infect humans due to their resistance to the cytotoxic action of HPR. Analysis of human infectious and non-infectious lines of T. b. rhodesiense resulted in the identification of a trypanosome gene what is necessary for human infection. The serum resistance associated (SRA) gone is highly conserved in T. b. rhodesiense isolates and is a member of the variant surface glycoprotein (VSG) superfamily. The SRA gene is absent in T. b. brucei, although similar gene sequences are present. To define the mechanism of human infectivity, we have selected for T. b. brucei lines resistant to TLF. The TLF resistant lines of T. b. brucei are able to avoid the cytotoxicity of HPR despite lacking the SRA gene. Examination of genome-wide gene expression patterns in TLF susceptible and resistant 7-.b. brucei lines will provide a coherent picture of the biological pathways in trypanosomes that influence host range and human infection. As the genome sequence of T. b. brucei is largely complete and we have been able to select for isogenic lines of T. b. brucei with differing degrees of susceptibility to TLF in vitro, we propose to utilize Serial Analysis of Gene Expression (SAGE) to monitor genome-wide levels of mRNA expression associated with human virulence. To detect regulation of genes related to human infectivity, we will perform SAGE by generating approximately 14,000 21 bp sequence tags from the mRNA of 10 isogenic lines of T. b. brucei (427 strain) differing in level of resistance to TLF. This research will provide a comprehensive understanding of changes in trypanosome gene expression in response to selective pressure of human innate immunity and will provide clues as to the origin of the SRA gene.